Telomerase is the ribonucleoprotein complex responsible for replicating the G-rich strand of telomeres, the physical ends of chromosomes. In humans, it is composed of a 451 nt RNA (hTR) that includes an RNA template, and associated proteins, including a unique reverse transcriptase (hTERT). In this grant application, we propose structural, thermodynamic, and functional studies of conserved domains of hTR and protein-RNA interactions that have been proposed to be important for telomerase activity, accumulation, and localization, and investigate the effect of telomerase RNA mutations linked to disease. Primarily NMR, as well as some X-ray crystallography, will be used for structural studies. The specific aims are: (1) Determine the structure of the highly conserved p2b-p3 pseudoknot sequence and the functional importance of the conserved nucleotides; 2) Determine the structure of the pseudoknot (core) domain of hTR by a combination of structural studies and modeling; (3) Investigate the possible interaction of the RT domain of hTERT with the pseudoknot; (4) Investigate the CR7 hairpin loop to determine the sequence and structural elements involved in Cajal body localization and in accumulation of hTR; (5) Determine the structure of the telomeras 3. H/ACA domain and investigate its interactions with the human H/ACA snoRNP proteins NopIO, Nh2, Gar1, and dyskerin. In spite of the high level of interest in telomerase and its biological and medical importance, almost no three-dimensional structural studies have been reported to date. No structures of telomerase proteins have been determined, either alone or in complex with the TR. The overall objective of this work is to provide a structural basis for understanding some of the RNA and RNA-protein interactions that occur in the functional telomerase ribonucleoprotein complex. The results of these investigations will provide a molecular basis for understanding telomerase function as well as mutations linked to disease, and provide a rational basis for design of drugs to affect telomerase activity in vivo.